KR20170021351A - Antitumor composition based on hyaluronic acid and inorganic nanoparticles, method of preparation thereof and use thereof - Google Patents

Abstract

The present invention relates to antitumor compositions comprising hydrophobized hyaluronan and inorganic nanoparticles stabilized by oleic acid. The hydrophilized hyaluronan in acylated hyaluronan form is provided as a carrier for inorganic nanoparticles in the composition. The composition may include super-magnetic nanoparticles, ZnO nanoparticles and up-converted nanoparticles among the inorganic nanoparticles. This composition selectively exhibits cytotoxicity against tumor cell lines of suspensions and adherent tumor cell lines, particularly colon carcinomas and adenocarcinomas, lung carcinomas, hepatocellular carcinomas and breast adenocarcinomas. The maximal cytotoxic effect was observed in a composition comprising oleyl derivative of hyaluronan and SPION. Also, compositions comprising acylated hyaluronan and SPION can also be advantageously used to detect in vivo in the body, preferably by accumulation of tumors or compositions in the liver. The composition can be sterilized in the form of a final product.

Description

TECHNICAL FIELD The present invention relates to an antitumor composition based on hyaluronic acid and inorganic nanoparticles, a method for producing the antitumor composition, and a method for producing the antitumor composition.

The present invention relates to compositions based on hyaluronic acid which can be used to treat tumor diseases. The composition comprises a hydrophobized derivative of hyaluronic acid, or a pharmaceutically acceptable salt thereof, and a nanoparticle stabilized with oleic acid, preferably a supernatant iron nanoparticle, a zinc nanoparticle or an upconversion and polymeric nanomicell, including up-conversion nanoparticles.

For the treatment of tumor diseases, chemotherapy is most commonly used to administer a drug substance, a system that is dispersed throughout the body, intravenously or orally to a patient. However, antitumor agents are very harmful to tumor cells as well as healthy cells. Thus, as a result of systemic dispersion, antitumor agents become toxic not only in the site where the tumor disease occurred, but also in the healthy tissue and cell area. Moreover, the non-selective dispersion of the drug reduces the amount of the drug substance eventually reaching the tumor cells, thereby reducing the efficacy of the treatment.

Due to the above-mentioned aspects, there is a great deal of interest in finding suitable strategies to enhance the efficacy of chemotherapy on tumor cells while at the same time inhibiting the inadequate systemic toxicity of therapeutic agents. When the drug is incorporated into the matrix of the carrier system, it has been found that the adverse side effects of the drug substance are significantly inhibited, and sometimes even erased. For this purpose, the research aims to target the carrier system to tumor cells.

Today, there are two most common strategies for targeting. One of them is based on so-called passive targeting, in which permeability and retention enhancement of tumor tissue is applied (so-called EPR effect) (Maeda, 2001). Tumor cells, as opposed to healthy tissues, are characterized by a perforated vascular structure that allows nanometer-sized molecules to be taken from the bloodstream into the tumor. However, manual targeting, in addition to other factors, has somewhat non-efficient and non-specific limits for tumor cells to capture carrier systems with drug substances (Duncan & Gaspar, 2011; El-Dakdouki, Pure & Huang, 2013). However, if the carrier system has a size suitable for manual targeting, this targeting method may combine the antitumor effect enhancement of the therapeutic agent with tumor cells due to other properties of the carrier. For example, patent application WO2008 / 130121 claims a tumor-selective biodegradable cyclotriphosphazene-platinum (II) conjugate that forms polymeric micelles in aqueous solution, which is useful for tumor tissue as compared to similar conjugates of US2001 / 6333422 Enhance selectivity in manual targeting. The selective enhancement of tumor cells in the manual targeting is also effected by the drug substance (SN-38, PI-103, etoposide, phenretinide) bound by the ester phenol bond cleaved rapidly and retinoic acid Eta < / RTI > conjugates, which are incorporated herein by reference. In both cases, the selectivity enhancement was detected based on the experimental data when compared to a similar system.

Another way to make the targeting, or selective, action of the drug more effective is when the therapeutic agent is modified by a high affinity ligand to a receptor located on the surface of tumor cells (Duncan & Gaspar, 2011; Ruoslahti, Bhatia & Sailor , 2010). The second strategy, called active targeting, should ensure selective treatment of tumor cells. As an example, the solution disclosed in US2007 / 0155807 can be exemplified, claiming a carboxylic acid derivative of thiazolidinone amide and thiazolidinamide, wherein selective action on melanoma cells with increased LPL receptor expression is detected . The problem with this and similar solutions is that LPL receptors are expressed in healthy cells, such as myocardial cells, as well, and can also act selectively in healthy tissues. Another problem with this solution and other similar solutions (e.g., WO 2012/173677, US 2013/02742200) is that the expression of tumor cell receptors is variable in time and patient (Duncan & Gaspar, 2011). The bound ligand selected for active targeting may be effective only in cells at certain stages of the tumor disease, or may be effective only in some patients.

Another possibility of active targeting is to target the carrier system using an external magnetic field when the system comprises magnetic nanoparticles bound in covalent or non-covalent association. In this case, a supernatant nanoparticle (SPION) may preferably be used. Most commonly, this is an Fe ₃ O ₄ nanoparticle considered as an MRI inactive imaging means without any intended pharmacological function (Huang et al., 2013). So far no short-term or long-term toxicity has been reported for intracellular internalization in relation to SPION (Huang et al., 2013). SPION can be used as a carrier in addition to MRI contrast and magnetic targeting, as a cell proliferation inhibitor or in combination with other pharmaceutical substances. Another preferred use is magnetic fluid hyperthermia where SPION absorbs alternating magnetic field energy and converts it into heat. Therefore, it is possible to selectively raise the temperature of the region where the SPION is located. If SPION is located in the tumor area, tumor cells can be destroyed using warmed temperatures because tumor cells are much more sensitive to temperature than healthy cells (Laurent, Dutz, Hafeli & Mahmoudi, 2011).

To enhance the selectivity of therapeutic agents, several distinctive characteristics of tumor cells compared with healthy cells are used in combination with manual targeting or active targeting (Fleige, Quadir & Haag, 2012). Such properties include, for example, higher acidic pH or increased concentration of reactive oxygen species (ROS). The patent application US2013 / 0230542 discloses a therapeutic component (phenol derivative) that is activated in the presence of ROS, i.e. it will selectively function in tumor cells with increased ROS concentration. The problem with this solution is that the increase in ROS concentration occurs in certain healthy cells as well. An example is a macrophage, where pathogen clearance occurs in the phagosome due to high levels of ROS. In addition, increased ROS concentration functions as a natural defense mechanism against hypoxia in other cells and also as a signaling molecule that acts on many physiological functions.

According to the literature, there are anticancer compositions that are expected to act antitumorally on some types of tumor cells in vitro. Examples thereof are the compositions of US 2005/0255173 comprising 1 to 3 components selected from the group consisting of: citric acid, zinc and albumin. This composition was more cytotoxic in vitro to human cell lines derived from adenocarcinomas NIH: OV-CAR-3 and SKOV-3 than control cells WI38 (normal human lung fibroblast). A disadvantage of this composition is that its antitumor effect is determined by the concentration of each component (US 2005/0255173). As the component is a body-innate material, the effect of the claimed composition may be influenced by the local concentration of the individual components in the provided administration site (e. G., High concentrations of albumin in the blood).

According to the cited documents, research on antitumor agents has been carried out all over the world. However, the clinical success of the proposed solution is still challenging, especially because most of the solutions contain organic polymers that are not biodegradable and do not have sufficient selectivity for tumor cells. For this reason, there is a continuing interest in discovering new compositions that have selective effects on tumor cells.

Duncan, R., & Gaspar, R. (2011). Nanomedicine (s) under the Microscope. Molecular Pharmaceutics, 8 (6), 2101-2141. El-Dakdouki, M. H., Pure, E., & Huang, X. (2013). Development of drug loaded nanoparticles for tumor targeting. Part 1: synthesis, characterization, and biological evaluation in 2D cell cultures. Nanoscale, 5 (9), 3895-3903. El-Dakdouki, M. H., Zhu, D. C., El-Boubbou, K., Kamat, M., Chen, J., Li, W., & Huang, X. (2012). Development of Multifunctional Hyaluronan-Coated Nanoparticles for Imaging and Drug Delivery to Cancer Cells. Biomacromolecules, 13 (4), 1144-1151. Fleige, E., Quadir, M.A., & Haag, R. (2012). Stimuli-responsive polymeric nanocarriers for the control of active compounds: Concepts and applications. Advanced Drug Delivery Reviews, 64 (9), 866-884. Huang, G., Chen, H., Dong, Y., Luo, X., Yu, H., Moore, Z., Bey, E. A., Boothman, D. A., & Gao, J. (2013). Superparamagnetic iron oxide nanoparticles: amplifying ROS stress to improve anticancer drug efficacy. Theranostics, 3 (2), 116-126. Laurent, S., Dutz, S., Hafeli, U. O., & Mahmoudi, M. (2011). Magnetic fluid hyperthermia: focus on superparamagnetic iron oxide nanoparticles. Advances in Colloid and Interface Science, 166 (1-2), 8-23. Li, M., Kim, H. S., Tian, L., Yu, M. K., Jon, S., & Moon, W. K. (2012). Comparison of Two Ultrasmall Superparamagnetic Iron Oxides on Cytotoxicity and MR Imaging of Tumors. Theranostics, 2 (1), 76-85. Maeda, H. (2001). The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting. Advances in Enzyme Regulation, 41 (1), 189-207. Ruoslahti, E., Bhatia, S. N., & Sailor, M. J. (2010). Targeting of drugs and nanoparticles to tumors. The Journal of Cell Biology, 188 (6), 759-768.

The present invention relates to hyaluronan nano micelles in combination with inorganic nanoparticles as selective antitumor agents. More particularly, the present invention relates to a composition based on hydrophobized hyaluronic acid and inorganic nanoparticles that selectively acts on cells derived from colorectal adenocarcinoma or adenocarcinoma, lung carcinoma, hepatocellular carcinoma and breast carcinoma. This composition can also be used as an in vivo imaging medium. Further, the present invention relates to a process for producing the composition.

The present compositions are based on loading inorganic nanoparticles stabilized with oleic acid in hydrophobized hyaluronan nano-micelles. The mounting can be performed by ultrasonication of the nanoparticle solution in an organic solvent with a hydrophobized hyaluronan aqueous solution. The prepared nanomyelite containing inorganic nanoparticles can then be separated from the free inorganic nanoparticles by centrifugation and used for selective effect on tumor cells. A major and unique advantage of the compositions of the present invention is that they exhibit selective in vitro activity against tumor cells, even when treated with a mixture of tumor cells and control cells. The composition comprises inorganic nanoparticles stabilized by oleic acid, wherein the original purpose of the inorganic nanoparticles was to enable in vivo detection of the composition after administration to the body. Surprisingly, however, when the composition is combined with an oleoyl derivative of hydrophobized hyaluronic acid, particularly hyaluronic acid, it is possible to selectively induce cytotoxicity in vitro in tumor cells without any cell proliferation inhibitor or other therapeutic substance . Unexpected and unexplained effects have been observed in SPION nanoparticles, zinc oxide nanoparticles, and up-converted nanoparticles, which are stabilized by oleic acid. Unexpected effects can not be clearly accounted for as receptor-mediated effects mediated through hyaluronan-mediated CD44 receptors, but also correlate observed selectivity with data collected so far to the effect of increasing ROS production I can not. However, selectivity can be induced by another intracellular release mechanism of the nanoparticle ion. The selective effect on tumor cells is rather surprising in that SPION incorporated into polysaccharide or other polymer matrix based carriers is used to be interpreted as non-cytotoxic (El-Dakdouki et al., 2012; Li, Kim, Tian, Yu, Jon & Moon, 2012).

Other advantages of the present compositions include compatibility with physiological solutions, the possibility of intravenous administration in vivo, and nanomyelite stability during the appropriate time at physiological pH. Another advantage of this composition is the use of hyaluronan as a carrier polymer to form an outer membrane surrounding the nano-micelle system, thereby ensuring the in vivo suitability of the composition. Preferably, the hyaluronan can assist in binding the composition to tumor cells characterized by increased CD44 receptor expression. The presence of SPION in the composition can be preferably used to target the carrier to a desired location in the body using a magnetic field. Alternating magnetic fields can be used to induce hyperthermia, leading to the destruction of tumor tissue. Another advantage is that a given composition can be combined with other active substances such as cell division inhibitors. Like SPION and zinc oxide nanoparticles, the presence of up-converted nanoparticles can also be used to detect in vivo compositions, preferably in tissue. In addition, up-converted nanoparticles with a particular composition can be used to control-release the drug from photodynamic therapy or composition. The presence of zinc oxide nano-particles preferably are used in a higher acidic pH tumor tissue, here ZnO nanoparticles are dissolved shows a local cytotoxicity in emitting Zn ^{2 +} ions and a high concentration region.

Thus, the present invention relates to a process for the preparation of a pharmaceutical composition comprising an acylated hyaluronan and an inorganic nanoparticle stabilized by oleic acid and selected from the group consisting of a super-magnetic nanoparticle, an up-converted nanoparticle or a zinc oxide nanoparticle, ≪ / RTI > Acylated hyaluronan is a C ₆ -C ₁₈ -acylated derivative of hyaluronic acid with saturated and unsaturated bonds, especially C _{18: 1} of hyaluronic acid Acylated derivatives, and acylated hyaluronan is used as a carrier of inorganic nanoparticles. When the composition according to the present invention comprises superpowder nanoparticles, the nanoparticles are preferably nanoparticles of iron oxide based on 0.3-3 wt.%, Preferably 1.0 wt.% Fe in the composition. to be. The size of the superpowder magnetic nanoparticles is 5 to 20 nm, preferably 5-7 nm, more preferably 5 nm. When the antitumor composition comprises zinc oxide nanoparticles, the content of Zn in the composition is preferably in the range of 0.3 - 3% by weight. When the antitumor composition comprises up-converted nanoparticles, it is preferably present in an amount such that the total amount of rare earth elements in the composition is between 0.3 and 3% hm. The up-converted nanoparticles may include, for example, Er, Yb and Y. [ The advantage of the composition according to the invention is that it can also be sterilized in the form of a final product using automatic sterilization.

The antineoplastic composition according to the present invention inhibits the proliferation of both adhesion and suspension human tumor cell lines derived from colorectal carcinomas and adenocarcinomas, lung carcinomas, hepatocellular carcinomas, breast carcinomas, preferably colorectal carcinomas and adenocarcinomas Lt; / RTI > Furthermore, antitumor compositions comprising superparamagnetic nanoparticles can be used as in vivo contrast agents, i.e., to detect accumulation of the composition in the body, particularly the liver and lesion development, e.g., a tumor. The compositions according to the present invention have been shown to exhibit different patterns of metal ion release in tumor and non-tumor cells, particularly in cells derived from human colorectal adenocarcinoma (= tumor) and human dermal fibroblast (= non-tumor) .

The anti-tumor composition according to the present invention can be applied as parenteral or topical administration, for example, for intravenous administration. Other additives used in the pharmaceutical composition may be further included, preferably sodium chloride, dextrose or buffering salt.

The composition according to the present invention can be prepared in the following manner: an aqueous solution of an acylated derivative of hyaluronic acid is prepared and then inorganic particles dispersed in a halide solvent such as chloroform are added and the resulting suspension is homogenized The inorganic nanoparticles are separated from the inorganic nanoparticles loaded on the nanomicels by centrifugation and subsequent filtration, after which the inorganic nanoparticles are stabilized by oleic acid, Nanoparticles, up-converted nanoparticles, or zinc oxide nanoparticles. The filtrate can then be lyophilized for long term storage or sterilized by automatic sterilization. The lyophilized product can then be dissolved in an aqueous solution and sterilized by automatic sterilization.

For the purposes of the present invention, commercially available SPION stabilized with oleic acid may be used.

Fig . TEM photograph of nanoparticles (SPION) surrounded by hydrophobized hyaluronan capsules.
2A, 2B, 2C . Inhibition of survival of compositions consisting of acylated hyaluronan and SPION in tumor HT-29 cell line versus positive / neutral effect in control NHDF fibroblast and mouse non-tumor 3T3 cell lines.
3 . The effect of inhibiting the viability of compositions composed of acylated hyaluronan and SPION in various tumor cell lines versus positive effects in control NHDF fibroblasts and mouse non-tumor 3T3 cell lines.
4A, 4B, 4C . Comparison of the effect of 5 nm, 10 nm, 20 nm SPION compositions encapsulated with acylated hyaluronan on survival of tumor cells HT-29 and healthy cells NHDF and 3T3.
5A, 5B, 5C . Survival inhibition by the composition of acylated hyaluronan and zinc nanoparticles in tumor HT-29 cell line versus positive / neutral effect in control NHDF fibroblast and mouse non-tumor 3T3 cell lines.
6A, 6B, 6C . Survival inhibition by acylated hyaluronan and up-converted nanoparticle composition in tumor HT-29 cell line versus positive / neutral effect in control NHDF fibroblast and mouse non-tumor 3T3 cell lines.
7 . Co-culture of healthy NHDF fibroblasts and control HT-29 cells with addition of acylated hyaluronan and SPION compositions.
Figure 8 . Expression of CD44 receptors on the surface of NHDF, MCF-7 and MDA-MB-231 cells as measured by flow cytometry.
Figure 9. Induction of ROS formation by compositions with acylated hyaluronan and SPION in NHDF and HT-29.
Figure 10. Intracellular Fe staining for tumor HT-29 and control NHDF cells after incubation with a composition of acylated hyaluronan and SPION (scale: 10 mu m).
11 . MRI detection (glioblastoma tumors, 1.1 mg Fe / kg) on the accumulation of SPION in tumors mounted in acylated hyaluronan after intravenous administration.
12 . Magnetic resonance angiography (1.1 mg Fe / kg) following intravenous administration of the SPION composition loaded in acylated hyaluronan.
13 . (2 hours and 24 hours after administration of the SPION composition) in tissue fragments after staining with Prussian blue.
FIG . Fe detection in hepatic tissue fragments after staining with Prussian blue (2 hours and 24 hours after SPION composition administration).
15. TEM photograph of nanoparticles (SPION) encapsulated in hydrophobized hyaluronan after sterilization treatment.
Figure 16. Selective cytotoxicity of SPION compositions before and after sterilization by automatic sterilization.
17A, 17B, 17C. Induction of cell suicide by SPION composition in mouse tumor lymphoma cell line EL4.

Example

SS = degree of substitution = 100% * molar amount of bonded substituent / molar amount of total polysaccharide dimer

As used herein, the term equivalence (eq) is to hyaluronic acid dimer unless otherwise stated. Percentages are percent by weight unless otherwise noted.

The molecular weight of hyaluronic acid (source: Contipro Pharma, a.s., Dolni Dobrouc, CZ) was measured by SEC-MALLS.

The term inorganic nanoparticle refers to an inorganic nanoparticle having a diagnostic function, and the diagnostic function is a common basic characteristic of the inorganic nanoparticles used in the composition according to the present invention. The diagnostic function is intended to mean the possibility of detecting these particles through methods available in medicine. SPION uses magnetic resonance, ZnO and up-converted nanoparticles can be detected using emission imaging, all of which are optimized for detection, which is why they are used. We chose to be able to detect in vivo or in vitro (micelles) in a set of inorganic nanoparticles.

The term up-conversion nanoparticles refers to nanoparticles containing up-converted lanthanoid nanoparticles, ie, other inorganic nanoparticles capable of efficient energy up-conversion, since they are not known.

Example  One Hydrophobicized  Preparation of an oleyl derivative of hyaluronic acid (C18: 1) using hyaluronic acid, especially a mixture of anhydrous benzoic acid and oleic acid.

100 g (250 mmol, 15 kDa) of sodium hyaluronan was dissolved in 2000 ml of demineralized water. Then 1000 ml of isopropanol was slowly added. TEA (70 ml, 3 eq.) And DMAP (1.52 g, 0.05 eq.) Were then added to this solution. At the same time, oleic acid (35.3 g, 0.5 eq) was dissolved in 1000 ml of isopropanol and TEA (70 ml, 3 eq.) And benzoyl chloride (14.4 ml, 0.5 eq.) Were added to this solution. After acid activation, the precipitate was filtered off with HA solution. The reaction was allowed to proceed at room temperature for 3 hours. The reaction mixture was then removed and diluted with 1000 ml of demineralised water to which 95 g of NaCl had been added. And precipitated using four times anhydrous isopropanol, and the acylated derivative was isolated from the reaction mixture. After decanting, the precipitate was repeatedly rinsed with isopropanol aqueous solution (85% vol.).

SS 13% (measured by NMR).

_{^{1 H NMR (D 2 O)}} : δ 0.88 (t, 3H, -CH 2 -C H 3), δ 1.22-1.35 (m, 20H, (-CH 2 -) 10), δ 1.60 (m, 2H, _{-C H 2 -CH 2 -CO-),} δ 2.0 (4H, (-CH 2 -) 2), δ 2.41 (t, 2H, -CH 2 -CO-), δ 5.41 (d, 2H, CH = CH)

This example describes a general synthetic method for hydrophobically-derivatized derivatives of hyaluronic acid. However, this process is not limited to oleyl derivatives. Details of the synthesis of hydrophobically-derivatized derivatives are given in patent application number CZ PV2012-842.

Example  2. Average 5 nm  Sized SPION  Produce

1.80 g of paricylate, 0.35 ml of oleic acid and 13.35 ml of 1-octadecene were placed in a 50 ml three-necked flask. The mixture was heated to 100 < 0 > C under vacuum and the volatiles were held for 30 minutes with evaporation. The mixture was then heated to 280 DEG C under a weak argon stream and held at this temperature for 60 minutes. The mixture was treated with argon bubbles while reacting at 280 ° C. After cooling to room temperature, acetone was added to the reaction mixture and the nanoparticles were separated by centrifugation. The precipitated SPION was rinsed four times with a hexane / acetone mixture (1: 4 to 1: 1 sequentially) and finally dispersed in toluene and stored at 4 [deg.] C under dark conditions.

Yield: 78%

Size of nanoparticles: 5.2 ± 0.8 nm (electron micrograph results).

Example  3. Average 10 nm  Sized SPION  Produce

1.80 g of paricylate, 0.35 ml of oleic acid and 13.35 ml of 1-octadecene were placed in a 50 ml three-necked flask. The mixture was heated slowly to 100 < 0 > C under vacuum and held for 30 minutes to evaporate the volatiles. The mixture was then heated to a boiling point (~ 317 ° C) under a weak argon flow and held at that temperature for 60 minutes. After cooling to room temperature, SPION was separated in the same manner as in Example 2.

Yield: 74%

Size of nanoparticles: 9.8 ± 0.5 nm (results of electron microscopy).

Example  4. Average 20 nm  Sized SPION  Produce

1.80 g of a paricylate, 0.35 ml of oleic acid, 5.34 ml of 1-octadecene and 6 g of n-docosane were placed in a 50 ml three-necked flask. The mixture was heated slowly to 100 < 0 > C under vacuum and held for 30 minutes to evaporate the volatiles. The mixture was then heated to 315 DEG C under a weak argon flow and held at that temperature for 60 minutes. After cooling to room temperature, SPION was separated in the same manner as in Example 2.

Yield: 56%

Size of nanoparticles: 21.1 ± 3.1 nm (result of electron micrograph)

Example  5. ZnO  Manufacture of nanoparticles

The zinc acetate dihydrate (1185.30 mg; 5.4 mmol) was placed in a 250 ml three-necked flask and dissolved in methanol (90 ml) at room temperature. Meanwhile, a solution of tetramethylammonium hydroxide (1622.91 mg; 8.96 mmol) in methanol (22.39 ml) was prepared in a two-necked flask. These two solutions were degassed (bath temperature 50 ° C, output 120 W) while bubbling with argon in an ultrasonic bath for 15 minutes. The methanol solution of zinc acetate was heated to reflux in an oil bath (bath temperature 60 占 폚). After addition of oleic acid (310 L, 0.99 mmol), the mixture was warmed to the boiling point (bath temperature 85 DEG C). A solution of tetramethylammonium hydroxide in methanol was heated under reflux (at a bath temperature of 75 ° C) and rapidly added to a three-necked flask containing zinc acetate and oleic acid. The reaction mixture was refluxed with stirring at a constant rate (600 rpm) and bubbled with argon for 2 minutes (bath temperature 85 ° C). The mixture was then diluted with methanol (90 ml) and cooled in an ice bath for 15 minutes. The cooled mixture was centrifuged for 15 minutes (4000 xg, 4 < 0 > C). The particles were rinsed with ethanol (3 x 25 ml) and each rinse step followed by centrifugation for 10 min (4000 xg, 25 [deg.] C). The particles were dispersed in chloroform (45 ml) and stored at 4 ° C under dark conditions.

Quantum efficiency of fluorescence: 34% (measured by relative method, reference material = norharman)

Size of nanoparticles: 3.4 ± 0.3 nm (results of electron micrograph)

Example  6. work- Conversion  Manufacture of nanoparticles

A molar amount corresponding to 1.60 mmol of yttrium (III) acetate, 0.36 mmol of ytterbium (III) acetate and 0.04 mmol of erbium (III) acetate was placed in a three-necked flask of 100 ml volume, octadodec- Oleic acid (12.0 ml) was added. The mixture was vigorously agitated (600 rpm) and the mixture was slowly heated to 80 ° C in an oil bath. At this temperature, the mixture was stirred under vacuum until it became totally clear and stirred for a further 90 minutes from the time it became clear. The mixture is filled with argon in a flask all, and cooling to room temperature under an argon atmosphere and then was added to NaOH (200 mg) and NH ₄ F (296.3 mg) solution in methanol (20 ml), then mixture became turbid. The mixture was stirred at room temperature overnight, and the methanol was slowly evaporated at 65 [deg.] C (oil bath). The flask with the mixture was then transferred to a heating mantle controlled by a PID-controller. The mixture was gradually evacuated and slowly heated to 112 < 0 > C under vacuum and degassed at this temperature for 30 minutes. The flask with the mixture was then charged with argon and heated to 305 DEG C at a rate of 2 DEG C / min under a slight argon flow under air reflux. The mixture was allowed to stand at 305 DEG C for 110 minutes, then taken out under heating conditions and allowed to cool naturally to room temperature.

The up-converted nanoparticles were precipitated from the reaction mixture using ethanol (twice the volume of the reaction mixture) and then separated by centrifugation (RCF 3000 xg; 10 min). The nanoparticles (precipitate) were dispersed in hexane (5 ml), precipitated with ethanol (10 ml) and then separated by centrifugation (RCF 3000 xg; 7 min). In this way, the nanoparticles were purified three times with a hexane / ethanol system and three times with a hexane / acetone system. Finally, the nanoparticles were dispersed in chloroform (10 ml) and stored at room temperature.

Nanoparticle composition (ICP-OES): NaYF ₄ : Yb / Er (80 mol% Y, 18 mol% Yb, 2 mol% Er

Organic component ratio (TGA): 7%

Size of nanoparticles (electron microscope): 34 ± 2 nm

Example  7. SPION and  Hyaluronic Capronyl  Derivatives ( HAC6 ) ≪ / RTI >

150 mg (HAC6, DS = 60%, Mw = 38 kDa) of the acylated derivative of hyaluronan prepared according to Example 1 was dissolved in 15 ml of demineralized water for 2 hours with constant stirring on a magnetic stirrer. SPION (stabilized by oleic acid, nanoparticle size: 5 nm) prepared according to Example 2 was transferred from the toluene medium to the chloroform medium.

The acylated hyaluronan solution was transferred to a rosette sonicator (RZ 1, volume: 25 ml) immersed in an ice bath. First, the solution was sonicated for 60 seconds (ultrasound parameter: 200 W, amplitude 65%, cycle 0.5 s and sonotrode S2). Also, CHCl ₃ 5 mg of SPION dispersed in 3 ml was gradually added to this solution (ultrasound parameter: 200 W, amplitude 85%, cycle 0.8 s and small note load S2). The homogenized suspension was further sonicated for 15 minutes (ultrasound parameters: amplitude 65%, cycle 0.5 s and small note load S2). The glassy nanoparticles were separated by repeated centrifugation (3 x 4500 RPM for 10 minutes), and the resulting supernatant containing the hyaluronan nano-micelles loaded with the nanoparticles was taken, filtered at 1.0 쨉 m, lyophilized Respectively.

Amount of Fe loaded (ICP measurement): 1.1% (wt.)

Example  8. Hyaluronic Caprylil  Derivatives ( HAC8 )Wow With SPION  ≪ / RTI >

150 mg (HAC8, DS = 22%, Mw = 20 kDa) of the acylated derivative of hyaluronan prepared according to Example 1 was dissolved in 15 ml of demineralized water for 2 hours with constant stirring on a magnetic stirrer. SPION (stabilized by oleic acid, nanoparticle size: 5 nm) prepared according to Example 2 was transferred from the toluene medium to the chloroform medium.

The acylated hyaluronan solution was transferred to a rosette sonicator (RZ 1, volume: 25 ml) immersed in an ice bath. First, the solution was sonicated for 60 seconds (ultrasound parameter: 200 W, amplitude 65%, period 0.5 s and small note load S2). Also, CHCl ₃ 5 mg of SPION dispersed in 3 ml was gradually added to this solution (ultrasound parameter: 200 W, amplitude 85%, cycle 0.8 s and small note load S2). The homogenized suspension was further sonicated for 15 minutes (ultrasound parameters: amplitude 65%, cycle 0.5 s and small note load S2). The glassy nanoparticles were separated by repeated centrifugation (3 x 4500 RPM for 10 minutes), and the resulting supernatant containing the hyaluronan nano-micelles loaded with the nanoparticles was taken, filtered at 1.0 쨉 m, lyophilized Respectively.

Amount of Fe loaded (ICP measurement): 1.1% (wt.)

Example  9. Hyaluronic Caprilin  Derivatives ( HAC10 )Wow With SPION  ≪ / RTI >

150 mg (HAC10, DS = 15%, Mw = 15 kDa) of the acylated derivative of hyaluronan prepared according to Example 1 was dissolved in 15 ml of demineralized water for 2 hours with constant stirring on a magnetic stirrer. SPION (stabilized by oleic acid, nanoparticle size: 5 nm) prepared according to Example 2 was transferred from the toluene medium to the chloroform medium.

The acylated hyaluronan solution was transferred to a rosette sonicator (RZ 1, volume: 25 ml) immersed in an ice bath. First, the solution was sonicated for 60 seconds (ultrasound parameter: 200 W, amplitude 65%, period 0.5 s and small note load S2). Also, CHCl ₃ 5 mg of SPION dispersed in 3 ml was gradually added to this solution (ultrasound parameter: 200 W, amplitude 85%, cycle 0.8 s and small note load S2). The homogenized suspension was further sonicated for 15 minutes (ultrasound parameters: amplitude 65%, cycle 0.5 s and small note load S2). The glassy nanoparticles were separated by repeated centrifugation (3 x 4500 RPM for 10 minutes), and the resulting supernatant containing the hyaluronan nano-micelles loaded with the nanoparticles was taken, filtered at 1.0 쨉 m, lyophilized Respectively.

Amount of Fe loaded (ICP measurement): 1.2% (wt.)

Example  10. Hyaluronic Palmy toil  Derivatives ( HAC16 )Wow With SPION  ≪ / RTI >

150 mg (HAC16, DS = 9%, Mw = 15 kDa) of the acylated derivative of hyaluronan prepared according to Example 1 was dissolved in 15 ml of demineralized water for 2 hours with constant stirring on a magnetic stirrer. SPION (stabilized by oleic acid, nanoparticle size: 5 nm) prepared according to Example 2 was transferred from the toluene medium to the chloroform medium.

The acylated hyaluronan solution was transferred to a rosette sonicator (RZ 1, volume: 25 ml) immersed in an ice bath. First, the solution was sonicated for 60 seconds (ultrasound parameter: 200 W, amplitude 65%, period 0.5 s and small note load S2). Also, CHCl ₃ 5 mg of SPION dispersed in 3 ml was gradually added to this solution (ultrasound parameter: 200 W, amplitude 85%, cycle 0.8 s and small note load S2). The homogenized suspension was further sonicated for 15 minutes (ultrasound parameters: amplitude 65%, cycle 0.5 s and small note load S2). The glassy nanoparticles were separated by repeated centrifugation (3 x 4500 RPM for 10 minutes), and the resulting supernatant containing the hyaluronan nano-micelles loaded with the nanoparticles was taken, filtered at 1.0 쨉 m, lyophilized Respectively.

Amount of Fe loaded (ICP measurement): 1.2% (wt.)

Example  11. Hyaluronic acid Stearyl  Derivatives ( HAC18 )Wow With SPION  ≪ / RTI >

150 mg (HAC18, DS = 9%, Mw = 15 kDa) of the acylated derivative of hyaluronan prepared according to Example 1 was dissolved in 15 ml of demineralized water for 2 hours with constant stirring on a magnetic stirrer. SPION (stabilized by oleic acid, nanoparticle size: 5 nm) prepared according to Example 2 was transferred from the toluene medium to the chloroform medium.

The acylated hyaluronan solution was transferred to a rosette sonicator (RZ 1, volume: 25 ml) immersed in an ice bath. First, the solution was sonicated for 60 seconds (ultrasound parameter: 200 W, amplitude 65%, period 0.5 s and small note load S2). Also, CHCl ₃ 5 mg of SPION dispersed in 3 ml was gradually added to this solution (ultrasound parameter: 200 W, amplitude 85%, cycle 0.8 s and small note load S2). The homogenized suspension was further sonicated for 15 minutes (ultrasound parameters: amplitude 65%, cycle 0.5 s and small note load S2). The glassy nanoparticles were separated by repeated centrifugation (3 x 4500 RPM for 10 minutes), and the resulting supernatant containing the hyaluronan nano-micelles loaded with the nanoparticles was taken, filtered at 1.0 쨉 m, lyophilized Respectively.

Amount of Fe loaded (ICP measurement): 1.0% (wt.)

Example  12. Hyaluronic Ole  Derivatives ( HAC18: 1) and With SPION  ≪ / RTI >

150 mg (HAC18: 1, DS = 12%, Mw = 15 kDa) of the acylated derivative of hyaluronan prepared according to Example 1 was dissolved in 15 ml of demineralized water for 2 hours while being constantly stirred on a magnetic stirrer. SPION (stabilized by oleic acid, nanoparticle size: 5 nm) prepared according to Example 2 was transferred from the toluene medium to the chloroform medium.

The acylated hyaluronan solution was transferred to a rosette sonicator (RZ 1, volume: 25 ml) immersed in an ice bath. First, the solution was sonicated for 60 seconds (ultrasound parameter: 200 W, amplitude 65%, period 0.5 s and small note load S2). In addition, 5 mg of SPION dispersed in 3 ml of CHCl ₃ was gradually added to the solution (ultrasound parameter: 200 W, amplitude 85%, cycle 0.8 s and small note load S2). The homogenized suspension was further sonicated for 15 minutes (ultrasound parameters: amplitude 65%, cycle 0.5 s and small note load S2). The glassy nanoparticles were separated by repeated centrifugation (3 x 4500 RPM for 10 minutes), and the resulting supernatant containing the hyaluronan nano-micelles loaded with the nanoparticles was taken, filtered at 1.0 쨉 m, lyophilized Respectively.

Amount of Fe loaded (ICP measurement): 1.0% (wt.)

The form of nano-particles in the polymer micelle cluster is shown in Figure 1;

Example  13. Hyaluronic Ole  Derivatives ( HAC18: 1) and With SPION  ≪ / RTI >

120 mg (HAC 18: 1, DS = 12%, Mw = 15 kDa) of the acylated derivative of hyaluronan prepared according to Example 1 was dissolved in 12 ml of demineralized water for 2 hours with constant stirring on a magnetic stirrer. SPION (stabilized by oleic acid, nanoparticle size: 5 nm) prepared according to Example 2 was transferred from the toluene medium to the chloroform medium.

The acylated hyaluronan solution was transferred to a rosette sonicator (RZ 1, volume: 25 ml) immersed in an ice bath. First, the solution was sonicated for 60 seconds (ultrasound parameter: 200 W, amplitude 65%, period 0.5 s and small note load S2). Also, CHCl ₃ 7.25 mg of SPION dispersed in 4 ml was gradually added to this solution (ultrasound parameter: 200 W, amplitude 85%, cycle 0.8 s and small note load S2). The homogenized suspension was further sonicated for 15 minutes (ultrasound parameters: amplitude 65%, cycle 0.5 s and small note load S2). The glassy nanoparticles were separated by repeated centrifugation (3 x 4500 RPM for 10 minutes), and the resulting supernatant containing the hyaluronan nano-micelles loaded with the nanoparticles was taken, filtered at 1.0 쨉 m, lyophilized Respectively.

Amount of Fe loaded (ICP measurement): 1.8% (wt.)

Example  14. Hyaluronic Linoleil  Derivatives ( HAC18: 2) and With SPION  ≪ / RTI >

150 mg (HAC18: 2, DS = 12%, Mw = 15 kDa) of the acylated derivative of hyaluronan prepared according to Example 1 was dissolved in 15 ml of demineralized water for 4 hours with constant stirring on a magnetic stirrer. SPION (stabilized by oleic acid, nanoparticle size: 5 nm) prepared according to Example 2 was transferred from the toluene medium to the chloroform medium.

The acylated hyaluronan solution was transferred to a rosette sonicator (RZ 1, volume: 25 ml) immersed in an ice bath. First, the solution was sonicated for 60 seconds (ultrasound parameter: 200 W, amplitude 65%, period 0.5 s and small note load S2). In addition, 5 mg of SPION dispersed in 3 ml of CHCl ₃ was gradually added to the solution (ultrasound parameter: 200 W, amplitude 85%, cycle 0.8 s and small note load S2). The homogenized suspension was further sonicated for 15 minutes (ultrasound parameters: amplitude 65%, cycle 0.5 s and small note load S2). The glassy nanoparticles were separated by repeated centrifugation (3 x 4500 RPM for 10 minutes), and the resulting supernatant containing the hyaluronan nano-micelles loaded with the nanoparticles was taken, filtered at 1.0 쨉 m, lyophilized Respectively.

Amount of Fe loaded (ICP measurement): 0.98% (wt.)

Example  15. Hyaluronic Linolenyl  Derivatives ( HAC18: 3) and With SPION  ≪ / RTI >

150 mg (HAC18: 3, DS = 3%, Mw = 15 kDa) of the acylated derivative of hyaluronan prepared according to Example 1 was dissolved in 15 ml of demineralized water for 4 hours with constant stirring on a magnetic stirrer. SPION (stabilized by oleic acid, nanoparticle size: 5 nm) prepared according to Example 2 was transferred from the toluene medium to the chloroform medium.

The acylated hyaluronan solution was transferred to a rosette sonicator (RZ 1, volume: 25 ml) immersed in an ice bath. First, the solution was sonicated for 60 seconds (ultrasound parameter: 200 W, amplitude 65%, period 0.5 s and small note load S2). In addition, 5 mg of SPION dispersed in 3 ml of CHCl ₃ was gradually added to the solution (ultrasound parameter: 200 W, amplitude 85%, cycle 0.8 s and small note load S2). The homogenized suspension was further sonicated for 15 minutes (ultrasound parameters: amplitude 65%, cycle 0.5 s and small note load S2). The glassy nanoparticles were separated by repeated centrifugation (3 x 4500 RPM for 10 minutes), and the resulting supernatant containing the hyaluronan nano-micelles loaded with the nanoparticles was taken, filtered at 1.0 쨉 m, lyophilized Respectively.

Amount of Fe loaded (ICP measurement): 1.0% (wt.)

Example  16. Hyaluronic Ole  Derivatives ( HAC18: 1) and With SPION  ≪ / RTI >

150 mg (HAC18: 1, DS = 12%, Mw = 15 kDa) of the acylated derivative of hyaluronan prepared according to Example 1 was dissolved in 15 ml of demineralized water for 2 hours with constant stirring on a magnetic stirrer. SPION (stabilized by oleic acid, nanoparticle size: 10 nm) prepared according to Example 3 was transferred from the toluene medium to the chloroform medium.

The acylated hyaluronan solution was transferred to a rosette sonicator (RZ 1, volume: 25 ml) immersed in an ice bath. First, the solution was sonicated for 60 seconds (ultrasound parameter: 200 W, amplitude 65%, period 0.5 s and small note load S2). In addition, 5 mg of SPION dispersed in 3 ml of CHCl ₃ was gradually added to the solution (ultrasound parameter: 200 W, amplitude 85%, cycle 0.8 s and small note load S2). The homogenized suspension was further sonicated for 15 minutes (ultrasound parameters: amplitude 65%, cycle 0.5 s and small note load S2). The glassy nanoparticles were separated by repeated centrifugation (3 x 4500 RPM for 10 minutes), and the resulting supernatant containing the hyaluronan nano-micelles loaded with the nanoparticles was taken, filtered at 1.0 쨉 m, lyophilized Respectively.

Amount of Fe loaded (ICP measurement): 0.4% (wt.)

Example  17. Hyaluronic Ole  Derivatives ( HAC18: 1) and With SPION  ≪ / RTI >

150 mg (HAC18: 1, DS = 12%, Mw = 15 kDa) of the acylated derivative of hyaluronan prepared according to Example 1 was dissolved in 15 ml of demineralized water for 2 hours with constant stirring on a magnetic stirrer. SPION (stabilized by oleic acid, nanoparticle size: 20 nm) prepared according to Example 4 was transferred from the toluene medium to the chloroform medium.

The acylated hyaluronan solution was transferred to a rosette sonicator (RZ 1, volume: 25 ml) immersed in an ice bath. First, the solution was sonicated for 60 seconds (ultrasound parameter: 200 W, amplitude 65%, period 0.5 s and small note load S2). In addition, 5 mg of SPION dispersed in 3 ml of CHCl ₃ was gradually added to the solution (ultrasound parameter: 200 W, amplitude 85%, cycle 0.8 s and small note load S2). The homogenized suspension was further sonicated for 15 minutes (ultrasound parameters: amplitude 65%, cycle 0.5 s and small note load S2). The glassy nanoparticles were separated by repeated centrifugation (3 x 4500 RPM for 10 minutes), and the resulting supernatant containing the hyaluronan nano-micelles loaded with the nanoparticles was taken, filtered at 1.0 쨉 m, lyophilized Respectively.

Amount of Fe loaded (ICP measurement): 1.7% (wt.)

Example  18. Hyaluronic Ole  Derivatives ( HAC18: 1 ), SPION  And Paclitaxel  ≪ / RTI >

150 mg (HAC18: 1, DS = 12%, Mw = 15 kDa) of the acylated derivative of hyaluronan prepared according to Example 1 was dissolved in 15 ml of demineralized water for 2 hours while being constantly stirred on a magnetic stirrer. Then, SPION 5 mg (stabilized by oleic acid, nanoparticle size: 5 nm) prepared according to Example 2 was transferred from toluene to chloroform. The nanoparticles thus prepared were mixed with 6 mg of paclitaxel dissolved in 3 ml of chloroform.

The acylated hyaluronan solution was transferred to a rosette sonicator (RZ 1, volume: 25 ml) immersed in an ice bath. First, the solution was sonicated for 60 seconds (ultrasound parameter: 200 W, amplitude 65%, period 0.5 s and small note load S2). Further, 5 mg of SPION and 6 mg of paclitaxel in CHCl ₃ were gradually added to this solution (ultrasound parameter: 200 W, amplitude 85%, cycle 0.8 s and small note load S2). The homogenized suspension was further sonicated for 15 minutes (ultrasound parameters: amplitude 65%, cycle 0.5 s and small note load S2). The free nanoparticles and paclitaxel were separated by repeated centrifugation (3 x 4500 RPM, 10 min), and the resulting supernatant containing the hyaluronan micelle loaded with the nanoparticles and paclitaxel was filtered off at 1.0 [mu] m, And lyophilized.

Amount of Fe loaded (ICP measurement): 1.5% (wt.)

Amount of PTX loaded (HPLC measurement): 0.3% (wt.)

Example  19. An olein derivative of hyaluronic acid ( HAC18: 1) and ZnO  Preparation of nanoparticulate compositions

150 mg (HAC18: 1, DS = 12%, Mw = 15 kDa) of the acylated derivative of hyaluronan prepared according to Example 1 was dissolved in 15 ml of demineralized water for 2 hours with constant stirring on a magnetic stirrer.

The acylated hyaluronan solution was transferred to a rosette sonicator (RZ 1, volume: 25 ml) immersed in an ice bath. First, the solution was sonicated for 60 seconds (ultrasound parameter: 200 W, amplitude 65%, period 0.5 s and small note load S2). Also, CHCl ₃ 5 mg of ZnO dispersed in 3 ml (Example 5) was gradually added to this solution (ultrasonic parameters: 200 W, amplitude 85%, period 0.8 s and soot roots S2). The homogenized suspension was further sonicated for 15 minutes (ultrasound parameters: amplitude 65%, cycle 0.5 s and small note load S2). The glassy nanoparticles were separated by repeated centrifugation (3 x 4500 RPM for 10 minutes), and the resulting supernatant containing the hyaluronan nano-micelles loaded with the nanoparticles was taken, filtered at 1.0 쨉 m, lyophilized Respectively.

Amount of Zn loaded (ICP measurement): 1.6% (wt.)

Example  20. An olein derivative of hyaluronic acid ( HAC18: 1) and  work- Conversion  Preparation of nanoparticulate compositions

150 mg (HAC18: 1, DS = 12%, Mw = 15 kDa) of the acylated derivative of hyaluronan prepared according to Example 1 was dissolved in 15 ml of demineralized water for 2 hours while being constantly stirred on a magnetic stirrer.

The acylated hyaluronan solution was transferred to a rosette sonicator (RZ 1, volume: 25 ml) immersed in an ice bath. First, the solution was sonicated for 60 seconds (ultrasound parameter: 200 W, amplitude 65%, period 0.5 s and small note load S2). Also, CHCl ₃ 5 mg (Example 6) of the up-converted nanoparticles SPION dispersed in 3 ml was gradually added to this solution (ultrasonic parameter: 200 W, amplitude 85%, period 0.8 s and small note load S2). The homogenized suspension was further sonicated for 15 minutes (ultrasound parameters: amplitude 65%, cycle 0.5 s and small note load S2). The glassy nanoparticles were separated by repeated centrifugation (3 x 4500 RPM for 10 minutes), and the resulting supernatant containing the hyaluronan nano-micelles loaded with the nanoparticles was taken, filtered at 1.0 쨉 m, lyophilized Respectively.

Er; Y; Yb payload (ICP measurement): 0.02; 0.50; 0.19% (wt.)

Example  21. Acylation Hyaluronan  And With SPION  Of the composition In vitro  Cytotoxicity

Primary cells, non-tumor cells and tumor cells (Table 1) were inoculated on 96-well panels and cultured for 24 hours at 37 ° C / 5% CO ₂ . Cells were then treated with the composition solutions of acylated hyaluronan and SPION obtained in Examples 7-14, 14 and 15 at concentrations of 10, 100, 200 and 500 / / ml (concentration of polymer micelle in the culture medium) Lt; / RTI > At the same time, survival (at each concentration) of acylated hyaluronan alone and SPION alone was measured. Viability of the cells was monitored at 0, 24, 48 and 72 hours using the MTT method, and the values obtained indicate inhibition or activation of cell survival at that time. The effects of various HAC18: 1 + SPION (Example 12) on the inhibition of cell viability ( Figure 2A-C ) and various tumor cell lines of cells treated with various acylated HA derivatives and SPION were monitored (Figure 3) . Figures 4A-C show comparative cytotoxic effects of compositions comprising SPION 5, 10 and 20 nm (Examples 12, 16, 17). The cell lines are shown in Table 1 .

Table 1. List of cell lines tested

Name of cell line Cell type / Origin Control cell line NHDF Invasive human dermal fibroblast 3T3 Mouse fibroblast Tumor cell line HT29 Human colon rectal adenocarcinoma A2058 Human melanoma A549 Human lung carcinoma C3A Human hepatocellular carcinoma MCF7 Human breast adenocarcinoma HCT116 Human colon rectal carcinoma MDA-MB231 Human breast adenocarcinoma Caco2 Human colon rectal adenocarcinoma

The results in Figures 2A-C show that, unlike the control cell lines (NHDF and 3T3), there is a significant inhibition of cell proliferation in the tumor cell line HT29. The maximal inhibition appeared in compositions based on C18 and C18: 1 acylated derivatives. The compositions containing C18: 1 derivatives and SPION as substrates showed stronger support for the viability of NHDF and 3T3 cells. Acylated hyaluronan alone and SPION did not affect survival in any test cell (data not shown).

The composition with HAC18: 1 and SPION was processed into other tumor cell lines ( Figure 3 ). The experimental data showed that the growth of tumor cell lines A549, HCT116, C3A, MCF7, MDA-MB231 and Caco2 was delayed. An exception was the melanoma cell line A2058, which showed only a slight inhibition when treated with the highest concentration (500 μg / ml) of the composition. Control fibroblasts (NHDF and 3T3), on the other hand, were markedly stimulated by the composition and did not exhibit any cytotoxic properties even at the highest concentration of 500 [mu] g / ml.

Figures 4A-C demonstrate selective anti-tumor activity of compositions comprising 5 nm SPION. Such an effect can not be observed at the above-mentioned levels in the composition containing 10 nm SPION and the composition containing 20 nm SPION.

Example  22. A composition comprising acylated hyaluronan and zinc oxide nanoparticles In vitro Cytotoxicity

Early human fibroblast (NHDF), intestinal tumor cells HT-29 and mouse fibroblast 3T3 were seeded in 96-well panels and cultured for 24 hours at 37 ° C / 5% CO ₂ . Then, the cells were treated with the composition solution of the acylated hyaluronan and zinc oxide nanoparticles obtained in Example 19 at a concentration of 10, 100, 200 and 500 占 퐂 / ml (concentration of polymer micelle). Cell viability was monitored at 0, 24, 48 and 72 hours using the MTT method, and the values obtained show inhibition or activation of cell survival at that time ( FIGS. 5A - C ).

The results in Figures 5A-C show that, unlike the control cell lines (NHDF and 3T3), the proliferation inhibition of tumor cell lines occurs at high concentrations of polymer micelles with increasing incubation time. However, at this time, proliferation inhibition was also observed in 3T3 cells treated with 500 ㎍ / ml. No inhibition was observed with lower concentrations of the composition and the control NHDF cell line.

Example  23. The composition of acylated hyaluronan and up-converted nanoparticles In vitro Cytotoxicity

Early human fibroblast (NHDF), intestinal tumor cells HT-29 and mouse fibroblast 3T3 were seeded in 96-well panels and cultured for 24 hours at 37 ° C / 5% CO ₂ . Cells were then treated with polymeric micellar solutions containing the up-converted nanoparticles obtained in Example 20 at concentrations of 10, 100, 200 and 500 占 퐂 / ml (concentration of polymer micelle). Cell viability was monitored at 0, 24, 48 and 72 hours using the MTT method, and the values obtained show inhibition or activation of cell survival at that time ( FIGS. 6A - C ).

The results in Figures 6A-C show that, unlike the highly viable control cell line NHDF, survival is inhibited in tumor cells as incubation time increases. However, some inhibition is also observed in 3T3, a non-tumor cell.

Example  24. Of a composition comprising acylated hyaluronan and SPION In vitro selective cytotoxicity

The early human fibroblasts labeled with DiO (green) and the tumor cells HT-29 labeled with DiI (red) were in a ratio of 3: 1 and were grown in RPMI 1640 (Roswell Park Memorial Institut) medium. After reaching a min 80% confluence of the cell monolayer, the cells were treated with 200 [mu] g / ml of the composition solution containing SPION of Example 12. After 72 hours of culture, cell photographs were taken using a fluorescence microscope Nikon Ti-Eclipse ( Fig. 7 ).

Expression of CD44 receptor on hyaluronan was analyzed by flow cytometry in NHDF, MCF-7 and MDA-MB-231 cells to elucidate possible differential active mechanisms for control and tumor cells. After reaching 80% confluence, the cells were rinsed with PBS and incubated with anti-CD44-FITC antibody for 15 minutes. When incubation was complete, the cells were rinsed twice with PBS and analyzed with a flow cytometer MACSQuant Analyzer (Miltenyi Biotec) . The results are expressed in terms of fluorescence intensity (RFU) ( Fig. 8 ).

In the case of NHDF cells and HT-29 cells, the oxidative stress was measured after treatment with the composition of acylated hyaluronan of Example 12 and SPION. Cells were cultured on a 6-well panel, and after reaching 80% confluence, 200 μg / ml of the composition solution containing SPION was treated for 24 hours. In the case of control cells, the test composition was not added and the medium was replaced once with fresh medium. After incubation, the cells were rinsed and treated with DCF-DA (non-fluorescent material oxidized by intracellular ROS to become fluorescent DCF, final concentration: 1 uM) for 20 min / 37 ° C / dark condition. Rinsed with PBS, and then the cells were analyzed on a flow cytometer MACSQuant Analyzer (Miltenyi Biotec). The results are expressed as the relative fluorescence intensity (% vs. non-treated control) of DCF within the cells ( Figure 9 ).

The results of FIG. 7 demonstrate that selective proliferation inhibition of tumor cell line HT-29 and, conversely, NHFF, a control fibroblast, do not adversely affect confluence. This effect was not caused by differential induction of ROS formation, and induction of ROS formation was increased and increased to the same level in both cell types (FIG. 9) . As an explanation for this, we can refer to differentiated levels of response to increased ROS production in NHDF and HT-29 cells.

Differences in activity against control cells and tumor cells are not associated with expression of the major surface receptor, hyaluronan, CD44. Figure 8 verifies that the expression level of the tumor cells MCF-7 and MDA-MB-231, which are highly expressed in the CD44 and high survival inhibition, is low in the control NHDF fibroblasts with increased viability by the composition 3).

Differential distribution of Fe ions was observed after staining cells incubated with the composition of acylated hyaluronan of Example 12 and SPION (presence of Fe using Prussian Blue), and in the tumor cells, dissolved Fe While iron aggregates were detected in control cells ( Figure 10 ). This phenomenon could be the cause of the selective activity of the composition in tumor cells.

Example 25. Preparation of a composition for intravenous administration

650 [mu] l of sterile 0.9% NaCl is added to 20-30 mg of the acylated hyaluronan and SPION composition of Example 12 prepared aseptically and the solution is stirred occasionally until the lyophilizate is completely dissolved. This solution is injectable in vivo without problems.

Solutions prepared in this manner are stable for at least 2 days as long as the hydrodynamic size of the particles.

Example 26. Preparation of a composition for intravenous administration

650 [mu] l of sterile 5% dextrose is added to 20-30 mg of the acylated hyaluronan and SPION of Example 12 prepared aseptically, and the solution is stirred occasionally until the lyophilizate is completely dissolved. This solution is injectable in vivo without problems.

Solutions prepared in this manner are stable for at least 2 days as long as the hydrodynamic size of the particles.

Example  27. Acylation Hyaluronan  And With SPION  In Vivo Detection of < RTI ID = 0.0 >

Lewis Brown Norweg rats with glioblastoma tumors were used for in vivo examination. This tumor was established by injecting 3 x 10 ⁶ glioma cells into the leg muscles and after 9 days the composition consisting of acylated hyaluronan (HAC18: 1) and SPION (750 μl of solution in 0.9% NaCl, Fe content 1.1 mg / kg) was intravenously administered. The rats were then analyzed using a Bruker Biospec (4.7 T).

Accumulation of SPION in the tumor after intravenous administration of the composition has been verified in Fig. 11, particularly where the dark portion is detected at the edge of the tumor. Visible accumulation of SPION was also detected in the liver ( Fig. 12 ), and thus this composition can be used as contrast medium in the liver.

The accumulation of SPION was also confirmed in tissue sections after tumor euthanasia ( FIG. 13 ) and liver ( FIG. 14 ), where the presence of Fe was detected by prussian blue staining (blue staining in the figure). No blue staining was detected in any of the control samples.

Example  28. Sterilization of autoclaved hyaluronan and SPION compositions

Sterilization of the composition prepared according to Example 12 (concentration: 30 mg / ml in 0.9% NaCl) was carried out in a sterilizer for 15 minutes at 121 占 폚.

The solution was stable after sterilization and the SPION remained clustered in hyaluronan nano-micelles ( Fig. 15 ), maintaining a selective cytotoxic effect on tumor cells.

Cytotoxicity was measured for tumor cell HT-29 and control primary NHDF fibroblasts according to the procedure described in Example 21. [ Figure 16 shows the results of comparing the composition of Example 12 before autoclaving and after sterilization, with selective cytotoxicity to tumor cells maintained after sterilization by automatic sterilization.

Example 29. Induction of cell suicide in mouse tumor-suspending lymphoma cell line EL4

The mouse lymphoma cell line EL4 (used for tumor induction in a mouse carcinogenicity test model) was cultured in RPMI 1640 (Roswell Park Memorial Institut) medium. In the exponential growth phase, aliquots were prepared from cell cultures at a concentration of 5 x 10 ⁵ cells / ml (RPMI medium), and the composition solution containing SPION of Example 9 was treated with 100, 200 and 500 μg / ml . After incubation for 72 hours, the cells were rinsed and developed specifically using a fluorescent marker (propidium iodide, Annexin V-FITC) for cell death and detected on a flow cytometer MACSQuant (Miltenyi Biotec).

In Figure 17A-C, Example 12 of SPION after 100 ㎍ / ml treatment a composition solution containing the apparent apoptotic induction (lower right quartile cell population, FIG. 17B) with some increase in the induction of cell death (left / upper right Quartile, Figure 17B). After the composition is treated at each concentration, live cells, apoptotic cells and necrotic cells are displayed on the graph (FIG. 17C).

Claims (23)

An antitumor composition comprising a C ₆ -C ₁₈ acylated derivative of hyaluronic acid according to general formula (I)
Further comprising inorganic nanoparticles containing stabilized oleic acid,
Wherein the inorganic nanoparticles are selected from the group consisting of a superpowder nanoparticle, an upconversion nanoparticle or a zinc oxide nanoparticle, and is selected from a superparamagnetic nanoparticle.

(I)
In this formula,
R is H ⁺ or Na ⁺ ;
R ¹ is H or -C (= O) C _x H _y or -C (= O) CH = CH -het, where x is an integer from the range 5-17, y is an integer in the range of 11-35, C _x H _y is a linear or branched, saturated or unsaturated C ₅ -C ₁₇ chain, and het is a heterocyclic or heteroaromatic moiety optionally containing N, S or O atoms;
In at least one repeat unit, one or more R < ¹ > -C (= O) C _x H _y or -C (= O) CH = CH-het;
n ranges from 12 to 4000. The method according to claim 1,
Wherein the acylated hyaluronan is a C ₆ -C ₁₈ acylated derivative of hyaluronic acid having a saturated bond and an unsaturated bond, in particular a C _{18: 1} acylated hyaluronic acid derivative. 3. The method according to claim 1 or 2,
Wherein the acylated hyaluronan is used as a carrier for the inorganic nanoparticles. 4. The method according to any one of claims 1 to 3,
Wherein the inorganic nanoparticles are superparamagnetic nanoparticles. 5. The method of claim 4,
Iron oxide based superpowder magnetic nanoparticles,
Wherein the Fe content in the composition is 0.3 - 3% wt., Preferably 1.0% wt. The method according to claim 4 or 5,
Wherein the nanoparticles comprise superparamagnetic nanoparticles having a size of 5-20 nm. The method according to claim 4 or 5,
An antitumor composition comprising superparamagnetic nanoparticles having a size of 5-7 nm. The method according to claim 4 or 5,
Lt; RTI ID = 0.0 > 5 nm < / RTI > size. 4. The method according to any one of claims 1 to 3,
Zinc oxide nanoparticles 0.3 - 3% wt. Lt; RTI ID = 0.0 > 1, < / RTI > 4. The method according to any one of claims 1 to 3,
Wherein the composition comprises up-converted nanoparticles in an amount such that the total content of rare earth elements in the composition is 0.3 - 3% wt. 11. The method of claim 10,
0.0 > Er, < / RTI > Yb, and Y. The anti- 12. The method according to any one of claims 1 to 11,
Wherein the composition is for use in inhibiting adherent growth and suspension growth of tumor cells. 13. The method according to any one of claims 1 to 12,
Wherein the composition is for use in inhibiting the proliferation of tumor cells derived from colorectal carcinomas and adenocarcinomas, lung carcinomas, hepatocellular carcinomas, breast adenocarcinomas, preferably from colorectal carcinomas and adenocarcinomas. 9. The method according to any one of claims 4 to 8,
Wherein said composition is for detecting in vivo accumulation of said composition in vivo, especially for use in detecting tumor and intracranial accumulation in vivo. 9. The method according to any one of claims 4 to 8,
Wherein the composition is for use in detecting pathological formation, particularly tumor formation, in vivo. 16. The method according to any one of claims 1 to 15,
Which is applicable as a parenteral administration or formulation for local administration. 17. The method according to any one of claims 1 to 16,
Further comprising other additives used in the pharmaceutical composition, preferably sodium chloride, dextrose or buffering salt. 18. The method according to any one of claims 1 to 17,
An antitumor composition, which further comprises a medical substance, preferably a cell growth inhibitor (cytostatic). 19. The method according to any one of claims 1 to 18,
An antitumor composition capable of being sterilized by a final casing by autoclaving. A process for the production of a composition according to any one of claims 1 to 19,
Preparing an aqueous solution of an acylated derivative of hyaluronic acid,
Adding inorganic particles dispersed in an organic halide solvent and stabilized by oleic acid;
Sonicating the formed suspension until a homogeneous mixture is formed; And
Separating the free inorganic nanoparticles from the nanomyelite-loaded nanoparticles through centrifugation and subsequent filtration,
Wherein the inorganic particles are selected from the group consisting of a super-magnetic nanoparticle, an up-converted nanoparticle or a zinc oxide nanoparticle. 21. The method of claim 20,
The filtrate is then lyophilized. 21. The method of claim 20,
The filtrate is then sterilized by automatic sterilization in the form of a final product. 22. The method of claim 21,
The lyophilisate is then dissolved in an aqueous solution and sterilized by automatic sterilization in the form of a final product.

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